Fig. 5

Metformin increases the number of tau inclusions in P301S mouse brain in vivo and induces aggregation of recombinant P301S tau in vitro. a Western blot analysis of tau in the sarkosyl-insoluble cortical fraction from P301S mice treated with or without metformin. The arrow indicates the bands of tau dimers. Tau was detected using the phosphorylation-and conformation dependent AT100 and Tau5 anti-tau monoclonal antibodies. MemCode was used to control equal loading. b-c Quantitative analysis of monomeric (b) and dimeric (c) tau in sarkosyl-insoluble fractions of P301S cortex. Densitometric values of tau bands are normalized over MemCode and bars represent the average ratio ± SEM. P301S, n = 4 / group. d. Representative images of MC1 immunoreactivity (d, d’; green) and FSB (d, d”; light blue) fluorescence in the cortex of P301S mice. Scale bars: in d, 50 μm; in d’-d”, 100 μm. e Representative images of AT8 immunoreactivity (e, e’; green) and FSB (e, e”; light blue) fluorescence in the cortex of P301S mice. Scale bars: in e, 50 μm; in e’-e”, 150 μm. f-g FSB⁺ cells in the prefrontal cortex (f) and hippocampus (g) of P301S mice. n = 3 / group. Bars represent the average number of FSB⁺ cells / mm² ± SEM. **p < 0.01, Student t-test. h-i. Gallyas silver staining in the cortex of P301S mice untreated (h) or treated with metformin (i). Scale bar 150 μm. j-k. Gallyas⁺ cells in the prefrontal cortex (j) and hippocampus (k) of P301S mice. n = 3 / group. Bars represent the average number of Gallyas⁺ cells / mm² ± SEM. **p < 0.01, Student t-test. l. Thioflavin T (ThT) fluorescence of recombinant P301S mutant human tau (P301Stau) aggregated in vitro in the absence (Ctr) or presence of 37.5 μM heparin (Hep) and/or 30 μM metformin (Met) for 2, 7 and 14 days. Data points represent the average ± SEM from at least 3 independent experiments. m-n. Representative negative staining electron microscopy image of fibrils of recombinant P301Stau aggregated in vitro for 7 days in presence of heparin (m) or metformin (n). In m, inset shows a high magnification of the squared area. In n, inset shows a high magnification image of tau aggregates. Scale bars: i-j, 2.5 μm; insets, 0.5 μm. o ThT fluorescence of recombinant P301Stau aggregated in vitro in the absence (control, Ctr) or presence of 37.5 μM heparin, 30 μM metformin, 30 μM phenformin (Phe) or 30 μM buformin (Buf) for 7 days. Fluorescence values are expressed as arbitrary units and normalized to the control. Bars represent the average ± SEM. **p < 0.01, one-way ANOVA followed by Holm-Sidak multiple comparison test. p Quantitative analysis of pTau in cortical neurons treated with or without 2.5 mM metformin, 30 μM phenformin and 30 μM buformin. pTau was detected using the AT8 monoclonal antibody and normalized on total tau detected by Tau5 antibody. Data are expressed as percentage of Ctr. Bars represent the average percentage ± SEM from 2 independent experiments. *p < 0.05, **p < 0.01, one-way ANOVA followed by Holm-Sidak multiple comparison test